Single molecule force spectroscopy (SMFS) is used to study the fundamental forces that drive the biological activity of cells and proteins. Nearly al of these biophysical measurements are performed in vitro using purified biomolecules. When studying the function and mechanics of cells, this approach yields inherently incomplete results by failing to account for the influence of the intracellular environment. Therefore, the development of a force spectroscopy system capable of in vivo sensing will advance biophysical measurement capability and help clarify the origins of the interactions driving cellular function. Broadly, a more complete understanding of cellular processes will aid researchers in the discovery and development of treatments for various disorders including cancer. This proposal outlines the development of a novel instrument that combines the manipulation capabilities of optical and magnetic tweezers to facilitate high resolution positioning as well as the application of a relatively large range of forces inside a living cell. To realize an instrumen of this type, an optical tweezers system will be retrofitted with a magnetic apparatus capable of generating controllable rotating magnetic fields. Magnetically anisotropic Janus spheres, less than 200 nm in diameter, will be fabricated to serve as probes that can be optically trapped and magnetically actuated. Their size will facilitate cellular uptake via endocytosis. Once inside the cell infrared laser beams or ultrafast laser pulses combined with external magnetic actuation will enable probe motility and force application without compromising the health of the cell. Advanced servo control schemes will allow the accurate application of forces and measurement of intracellular mechanics. Initially probes will be inserted into live granulocytes to access instrument performance. Once parameters are optimized the in vivo force spectroscopy instrument will be made available to the general research community for the characterization of a myriad of cell lines.